Introduction
Over the past 10 years, advancements in stem cell studies have highlighted potential applications of exogenous cell therapy in the treatment of various neurological diseases. Numerous animal and preclinical studies have shown that the transplantation of stem cells into damaged neural tissues can yield favorable results (Saracino et al., 2013; Gennai et al., 2015; Ge et al., 2016). In terms of cell types for clinical applications, mesenchymal stem cells (MSCs) are considered ideal candidates because they have self-renewal ability, multidirectional differentiation potential, no major ethical problems, and low immunogenicity (Bedini et al., 2018; Singh et al., 2020; Deng et al., 2022). Efforts to understand the biological behaviors of transplanted cells (e.g., distribution, proliferation, and differentiation) require a noninvasive method to monitor transplanted stem cells (Janowski et al., 2014; Dash et al., 2015; Ngen et al., 2015). Optical imaging, nuclear medicine imaging, and magnetic resonance imaging (MRI) are the main methods used for cell tracking (Silachev et al., 2016; Hwang et al., 2017; Namestnikova et al., 2017). Among these three imaging methods, MRI is ideal for clinical translation because it requires no radiation, has deep tissue penetration, and exhibits sufficient tissue contrast (Jurgielewicz et al., 2017; Ngen and Artemov, 2017).
Currently, two types of stem cell MRI tracking strategies are available: direct imaging (focused on intracellular magnetic labeling) and indirect imaging (focused on reporter genes). In direct imaging, the MRI signal produced by intracellular magnetic labeling can fade with stem cell proliferation, which hinders long-term cell tracking (Cai et al., 2007, 2008; Lee et al., 2014; Watada et al., 2015; Qin et al., 2018). The reporter gene-based imaging approach resolves this problem. Reporter genes can be integrated into the stem cell genome and sustainably expressed regardless of cell proliferation or differentiation; this leads to the accumulation of extracellular iron inside the cells, which facilitates MRI-based detection (Zheng et al., 2017). Thus far, many MRI reporter genes have been investigated; ferritin heavy chain 1 (FTH1) is most commonly studied because of its clear iron accumulation effect and safety (Vande Velde et al., 2013; He et al., 2016; Sun et al., 2021).
In our previous study, we transduced the FTH1 gene into MSCs, then conducted MRI analyses to longitudinally detect the transduced cells during proliferation and differentiation (Mu et al., 2018). We found that the FTH1 gene was clearly expressed in both stem cells and differentiated neuron-like cells (NLCs), which suggested that FTH1-based MRI could be used for long-term tracking of stem cells. However, this method cannot distinguish whether cells have undergone differentiation. Thus, it cannot be used to determine whether and/or when stem cells have differentiated into NLCs; this information is essential when evaluating cell fate. To solve this problem, we searched for a gene with distinct expression patterns in differentiated and undifferentiated cells. We hypothesized that we could detect the differentiation event in stem cells by using the promoter for the gene with differentiation-specific expression to activate reporter gene expression.
In this study, we added a neuron-specific enolase (NSE) promoter upstream of the FTH1 gene, then transduced the modified reporter gene into MSCs; we observed changes in FTH1 expression and the MRI signal during stem cell differentiation. Our approach of inducing FTH1 gene expression via the NSE promoter may facilitate MRI-based detection of stem cell differentiation into NLCs.
Methods
Ethics statement
Animal experiments in this study were approved by the Animal Ethics Committee of the Children’s Hospital of Chongqing Medical University, China, on January 20, 2021 (approval No. CHCMU-IACUC20210114). The experimental protocols and procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th ed) (National Institutes of Health, 2011). All possible efforts were made to alleviate animal suffering.
Preparation of MSCs
Bone marrow MSCs were obtained from two male Sprague-Dawley rats (grade: specific pathogen-free; age, 4 weeks; weight, ~50 g) from the Experimental Animal Center of Chongqing Medical University, Chongqing, China (license No. SCXK (Yu) 2018-0003). MSCs were isolated from bone marrow using a previously reported protocol (Cai et al., 2008). The isolated MSCs were grown in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (HyClone, Logan, UT, USA) at 37°C in 95% humidity with 5% CO2 . When MSCs reached 80–90% confluence, they were passaged by trypsinization. Third-passage MSCs were collected for experiments. To identify MSCs, cells were incubated with anti-rat antibodies to CD34, CD45, CD29, and CD44 (Beyotime, Nanjing, China) for 30 minutes in the dark, then transferred into 0.4 mL of phosphate-buffered saline (PBS); subsequently, they were sorted and quantified by flow cytometry.
Preparation of recombinant lentiviral vector
The upstream region of the NSE gene (NSE promoter, 1.8 kb) was amplified by polymerase chain reaction (PCR) using rat genomic DNA as a template, in accordance with previous reports (Peel and Klein, 2000). The following primers were used: forward, 5′-ATG CTC TAG ACT CGA GAG CTC TGA GCT CCT CCT CTG CTC GC-3′; reverse, 5′-CAT GGT GGC GAC CGG TTC GAG GAC TGC AGA CTC AGC CG-3′. The rat FTH1 gene (NM_012848) was synthesized by PCR using the following primers: forward, 5′-AGG TCG ACT CTA GAG GAT CCG GGC CCT ACG TGC TGT CTC ACA CAG CC-3′; and reverse, 5′-ACC GTA AGT TAT GTG CTA GCT TAT TTG TCG TCA TCA TCC TTA TAG TC-3′. FTH1 was inserted into the lentiviral vector through digestion with Eco RI and Bam HI to obtain the recombinant lentiviral vector pLV/CMV-FTH1. Then, the cytomegalovirus (CMV) promoter in pLV/CMV-FTH1 was replaced by the NSE promoter (NSE) using Cla I and Eco RI digestion to obtain pLV/NSE-FTH1, in which the NSE promoter controlled the expression of FTH1 (Figure 1A FTH1 gene and NSE promoter were confirmed by DNA sequencing and PCR. The recombinant vectors were confirmed by DNA sequencing, a double digestion system, and agarose gel electrophoresis. The pLV/NSE-FTH1 or pLV/CMV-FTH1 vectors and the packaging plasmids pMD2G and pSPAX2 (Hanbio Biotechnology Co., Ltd. Shanghai, China; Cat# LV011) were co-transfected into 293T packaging cells (Chinese Academy of Sciences, Beijing, China; Cat# GNHu 43; RRID: CVCL_0045) to obtain lentiviruses for transduction experiments. Six hours after transfection, fresh medium with 10% fetal bovine serum was supplemented; viral medium was collected 48 and 72 hours later. Centrifugation at 4000 × g was performed for 10 minutes at 4°C to remove cellular debris. Virus particles were collected by centrifugation at 82,700 × g for 2 hours at 4°C, then stored at –80°C.
Lentiviral transduction
For cell transduction, MSCs at 30–40% confluence were transduced with lentiviruses carrying LV/NSE-FTH1 or LV/CMV-FTH1, in the presence of 5 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). To determine the appropriate conditions for transduction, MSCs were transduced with lentiviruses at various multiplicities of infection (MOIs; 5, 10, 15, 20, or 30). MSCs treated with PBS were used as a control group. The expression patterns of green fluorescence protein were evaluated via fluorescence microscopy (Shanghai Piem Optical Instrument Co., Ltd., Shanghai, China) at 24, 48, and 72 hours after transduction to determine the optimal MOI. Puromycin (Sigma-Aldrich) at different concentrations (0.1, 0.5, 1, 2, 3, 4, or 5 μg/mL) was added to the transduced MSCs, then cultured for 7 days to determine the minimum lethal concentration. After treatment with puromycin at the minimum lethal dose for 7 days and then at half of the minimum lethal dose for 3 days, the resulting clonal cells were named MSCs/NSE-FTH1 and MSCs/CMV-FTH1.
Neuronal induction and assessment
To induce the cells (MSCs, MSCs/CMV-FTH1, and MSCs/NSE-FTH1) to undergo neuronal differentiation, a previously reported two-step neuronal induction protocol (Bi et al., 2010) was used. Briefly, stem cells were pre-induced for 24 hours by incubation with 1 mmol/mL all-trans retinoic acid (ATRA); they were induced for another 24 hours by incubation with modified neuronal induction medium (containing 200 μM butylated hydroxyanisole [Meilun Biological Products Co., Ltd., Dalian, China], 20 mM KCl [Sangon Biotechnology Co., Ltd., Shanghai, China], 8 μM forskolin [AmyJet Scientific Inc., Wuhan, China], 1.6 mM sodium valproate [Meilun Biological Products Co., Ltd.], 0.8 μM hydrocortisone [Meilun Biological Products Co., Ltd.], and 4 μg/mL insulin [Meilun Biological Products Co., Ltd.]). Cells subjected to neuronal induction in the three groups were designated NLCs, NLCs/CMV-FTH1, and NLCs/NSE-FTH1.
Before and after induction, the expression patterns of the neuronal markers NSE and microtubule-associated protein-2 (MAP-2) were evaluated by immunofluorescence. Dissociated cells (250 cells/mm2 ) were seeded onto coverslips and incubated for 12 hours; subsequently, they were fixed with 4% paraformaldehyde for 20 minutes, permeabilized in 0.02% Triton X-100, and then blocked with 5% bovine serum albumin (Sigma-Aldrich) for 1 hour. Next, the cells were incubated with the primary rabbit anti-NSE monoclonal antibody (1:150, Abcam, Cambridge, UK; Cat# ab180943; RRID: AB_11031988) or rabbit anti-MAP-2 monoclonal antibody (1:200, Proteintech, Chicago, IL, USA; Cat# 67015-1-Ig; RRID: AB_2882331) overnight at 4°C. Subsequently, the cells were washed with PBS and incubated with the appropriate goat anti-rabbit DyLight 594-conjugated secondary antibody (1:1000, Proteintech, Cat# rba594; RRID: AB_ 2631390) in the dark for 1 hour at 37°C. Then, the cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Proteintech) for 30 minutes. Immunofluorescence staining was observed under a fluorescence microscope (TE2000-S, Nikon, Tokyo, Japan).
The mRNA expression levels of NSE and MAP-2 before and after neuronal induction were measured via real-time PCR (RT-PCR). Total cellular RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA). Complementary DNA was obtained by reverse transcription of RNA with the PrimeScript RT Reagent Kit (TaKaRa, Tokyo, Japan). Using the synthesized complementary DNA as a template, PCR was conducted with the following primers: NSE forward, 5′-CCC ACT GAT CCT TCC CGA TAC AT-3′ and NSE reverse, 5′-CGA TCT GGT TGA CCT TGA GCA-3′; MAP-2 forward, 5′-CCA TTT GCA ACA GGA AGA CAC-3′ and MAP-2 reverse, 5′-CAG CTC AAA TGC TTT GCA ACT AT-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) forward, 5′-TTC AGC TCT GGG ATG ACC TT-3′ and GAPDH reverse, 5′-TGC CAC TCA GAA GAC TGT GG-3′. Real-time PCR amplification was conducted using a real-time PCR instrument (Funglyn Biotech, Shanghai, China). One microliter of cDNA was used as template in a 10-μL PCR reaction system that included 5 μL of PCR buffer (Western Biotechnology, Chongqing, China), 1 μL of forward and 1 μL of reverse primers, and 3 μL of diethyl pyrocarbonate-treated water (Sigma-Aldrich). The amplification conditions were as follows: 94°C for 4 minutes, followed by 30 cycles of 94°C for 20 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The mRNA expression levels of NSE and MAP-2 were determined using the 2–ΔΔCt calculation method (Xu et al., 2019), and the results were expressed as fold changes relative to the levels of GAPDH .
Evaluation of FTH1 gene expression
To evaluate the expression of FTH1 before and after neuronal induction of the transduced MSCs, western blotting was conducted in accordance with a previously reported protocol (He et al., 2015). Briefly, cells in each group were harvested and lysed with radioimmunoprecipitation assay buffer (Beyotime) and purified by centrifugation for 15 minutes (14,000 × g , 4°C). The total protein concentration was calculated using the bicinchoninic acid method (Beyotime) (Goldring, 2019), and the final concentration was adjusted to 5 g/µL. Each sample was mixed with sodium dodecyl sulfate loading buffer (1:4 ratio) and heated at 95°C for 5 minutes. Protein samples (30 μg each) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Madrid, Spain). After the membranes had been blocked with 5% bovine serum albumin in Tris-buffered saline containing Tween-20, they were incubated with rabbit anti-FTH1 antibody (1:1000; Abcam; Cat# ab183781; RRID: AB_11001434) or rabbit anti-GAPDH antibody (1:1000; Abcam; Cat# ab181602; RRID: AB_2630358) overnight at 4°C. After three washes in Tris-buffered saline containing Tween-20, the membranes were incubated with the horseradish peroxidase-tagged secondary antibody anti-rabbit (1:5000, Abgent, San Diego, CA, USA; Cat# ab11839c; RRID: AB_10819356) for 1 hour at 37°C; they were then visualized with a chemiluminescent detection reagent (Beyotime). The level of FTH1 expression was measured and standardized to the level of GAPDH expression. Additionally, the levels and patterns of FTH1 expression in cells before and after neuronal induction were assayed by immunofluorescence and RT-PCR, using the methods described above for NSE and MAP-2.
Effects of iron loading on cell viability and differentiation efficiency
Before evaluation of the iron transport efficiency with respect to FTH1 expression, ferric ammonium citrate (FAC) was added into the medium to provide iron for intracellular uptake. To determine the optimal concentration of FAC, 2 × 103 MSCs seeded in 96-well plates were treated with 1 × 100 , 1 × 101 , 1 × 102 , 2 × 102 , 5 × 102 , 1 × 103 , 2 × 103 , 5 × 103 , or 1 × 104 μM FAC for 72 hours; cell viability was then assayed. Cell viability and cell proliferation in the three groups (MSCs, MSCs/CMV-FTH1, and MSCs/NSE-FTH1) were evaluated at the optimal concentration of FAC. A Cell Counting Kit-8 method (Dojindo Laboratories, Kumamoto, Japan) was used, in accordance with the manufacturer’s protocol. Briefly, cells were cultured with 10% Cell Counting Kit-8 reagent for up to 4 hours; the optical density of the cells was then measured with a microplate reader (Thermo Fisher Scientific, New York, NY, USA) at a wavelength of 450 nm. The measurements were repeated three times.
Differentiation efficiency was also measured in the three groups (MSCs, MSCs/CMV-FTH1, and MSCs/NSE-FTH1). On images of NSE immunofluorescence, the numbers of NSE-positive and axon-forming cells were counted under 10 randomly selected fields of view. The proportion of these cells to all cells was regarded as the differentiation efficiency. Additionally, cell axon length was calculated using Quantity One 4.6 software (Bio-Rad, Hercules, CA, USA). All measurements were conducted three times independently.
Cellular MRI
After treatment with the optimal concentration of FAC, each group of cells (MSCs, MSCs/CMV-FTH1, MSCs/NSE-FTH1, NLCs, NLCs/CMV-FTH1, and NLCs/NSE-FTH1) was washed with PBS to eliminate free iron, then harvested by trypsinization and centrifugation (1000 r/min, 10 minutes). Cell phantoms for MRI were prepared by uniformly embedding 5 × 106 cells into 0.5 mL of 1% agarose in 600-μL Eppendorf tubes. A T2-weighted imaging (T2WI) sequence and a multi-echo sequence were performed on a 7.0 Tesla MRI scanning system (Bruker, Karlsruhe, Germany). The scanning parameters were set in accordance with our previously published protocol for cell phantom imaging (Sun et al., 2021). The multi-echo images were postprocessed to obtain T2 maps, which were used to measure R 2 values within a circular region of interest (ROI). The circular ROI in each group was drawn to cover the entire cell suspension at the largest possible diameter (~5 mm). Each measurement was repeated three times independently.
Intracellular iron detection
Accumulation of intracellular iron was qualitatively assessed via Prussian blue iron staining in accordance with a previously reported protocol (Cai et al., 2008). Briefly, cells grown on coverslips after 48 hours of treatment with FAC were thoroughly washed and fixed in 4% paraformaldehyde for 30 minutes before staining; the coverslips were then treated with Prussian blue staining solution (Beyotime) for 30 minutes. Cell nuclei were counterstained with Nucleoside red solution (Beyotime). The coverslips were observed by light microscopy (Nikon).
For quantification of intracellular iron content, 1 × 105 cells per group were subjected to iron measurement by atomic absorption spectrophotometry, in accordance with a previously reported protocol (Cao et al., 2018). At least three measurements were conducted for each sample. The mean iron content per cell is presented in picograms per cell.
In vivo experiments
Nine male Sprague-Dawley rats (age, 8 weeks; body weight, ~250 g) were randomly divided into three groups (n = 3/group) and subjected to intracerebral transplantation of MSCs, MSCs/CMV-FTH1, or MSCs/NSE-FTH1. For each rat, 5 × 105 cells were prepared by preinduction with 0.5 μM ATRA for 24 hours and suspension in 10 µL of modified neuronal induction medium. The rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg; Sigma-Aldrich), then positioned on a motorized stereotaxic frame (Beijing Jinyuan Science and Technology Co., Ltd., Beijing, China). After the rats’ heads had been shaved, a skin incision was made at the cranial midline, and a tiny hole was drilled 2 mm right of the striatum (bregma coordinates: 3 mm mediolateral, 0.4 mm anteroposterior, 4 mm dorsoventral). A microsyringe was gently and slowly inserted into the right brain parenchyma (striatum); the cell suspension was then injected over a period of 5 minutes. The syringe was maintained in this position for approximately 5 minutes; it was then carefully removed and the skin incision was sutured. To increase the iron concentration in brain tissue for enhanced cellular uptake, the rats were provided 5 mg/L of FAC in drinking water.
At 1, 3, and 7 days after cell transplantation, anesthetized rats were subjected to MRI examination. T2WI and multi-echo sequences were performed on a 7.0 Tesla MR scanning system. The T2WI scanning parameters were as follows: repetition time = 2500 ms, echo time = 35 ms, matrix = 380 × 300, field of view = 160 mm, and slice thickness = 1.2 mm. The multi-echo parameters were as follows: repetition time = 2000 ms and echo time = 8–200 ms with a step size of 8 ms (25-point T2 mapping); the matrix, field of view, and slice thickness were identical to the parameters used for T2WI. Based on the multi-echo imaging data, T2 maps were acquired and R2 values were measured within ROIs. The minimum ROI size was 50 pixels, covering all areas of low signal intensity in a blinded manner. Each measurement was repeated three times independently.
Immediately after MRI examination, the rats were deeply anesthetized with 3% pentobarbital sodium (Sigma-Aldrich), then sacrificed by asphyxiation with a high concentration of CO2 (95%, 5 L/min). The brains were extracted, fixed in 4% paraformaldehyde, dehydrated in alcohol and xylol, embedded in paraffin, and sliced into 4-μm-thick sections. The accumulation of iron in brain tissue was detected by Prussian blue staining. Additionally, iron content in brain tissue was quantified using the method described above. Three measurements were conducted for each sample, and the mean iron content was expressed in milligrams per gram.
Statistical analysis
No statistical methods were used to predetermine sample sizes; however, our sample sizes were similar to the numbers of animals used in a previous study (Minn et al., 2015). No rats were excluded from this analysis. Each test was repeated at least three times; the quantitative data are shown as means ± standard deviations (SDs). These data were analyzed using GraphPad Prism 5.01 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA). Student’s t -test was used for comparisons between experimental and control groups. Multiple group comparisons were performed using one-way analysis of variance, followed by Tukey’s post hoc test. P -values < 0.05 were considered statistically significant.
Results
Lentiviral construction and transduction
To ensure FTH1 was expressed only when MSCs differentiated into NLCs, FTH1 expression was controlled by the NSE promoter. PCR confirmed that the length of the recombinant FTH1 and NSE mRNA construct was between 2000 and 3000 bp (Figure 1B FTH1 gene and NSE promoter were correct (Additional file 1 Figure 1C
Figure 1: Lentiviral construction and mesenchymal stem cell transduction .(A) FTH1 was inserted into the MCS of pHBLV/CMV-MCS-3flag-EF1-ZSgreen-puro (pLV/CMV) by digestion with Eco RI and Bam HI to obtain pLV/CMV-FTH1. The NSE promoter replaced the CMV promoter in pLV/CMV-FTH1 by digestion with Cla I and Eco RI; this yielded pLV/NSE-FTH1, in which FTH1 expression was driven by the NSE promoter. (B) Verification of the recombinant FTH1 and NSE mRNA construct by PCR. The length of the recombinant construct was 2000–3000 bp. (C) Expression of GFP (green) was observed in MSCs/CMV-FTH1 and MSCs/NSE-FTH1. Scale bars: 50 μm. CMV: Cytomegalovirus; FTH1: ferritin heavy chain 1; GFP: green fluorescent protein; MCS: multiple cloning sites; MSCs: mesenchymal stem cells; MSCs/CMV-FTH1: ferritin heavy chain 1 overexpression triggered by cytomegalovirus promoter in mesenchymal stem cells; MSCs/NSE-FTH1: ferritin heavy chain 1 overexpression triggered by neuron-specific enolase promoter in mesenchymal stem cells;NSE: neuron-specific enolase; pLV/CMV: empty lentiviral vector plasmid containing cytomegalovirus promoter; pLV/CMV-FTH1: lentiviral vector plasmid containing ferritin heavy chain 1 gene triggered by the cytomegalovirus promoter; pLV/NSE-FTH1: lentiviral vector plasmid containing ferritin heavy chain 1 gene triggered by the neuron-specific enolase promoter.
Additional file 1
Identification of MSCs and NLCs
Microscopy showed that MSCs had a triangular or spindle-like morphology (Figure 2A Figure 2B
Figure 2: Identification of MSCs and NLCs .(A) Cell morphology analysis showed that the MSCs were triangular or spindle-shaped, with a distribution similar to the distribution in fish (Ranjbaran et al., 2018). After neuronal induction, the cells exhibited neuron-like morphological features, including the formation of long and thin processes, cytoplasmic contraction, and enhanced transparency. Arrows indicate neuron-like cells. Scale bars: 10 μm. (B) Flow cytometry analysis revealed that the expression levels of CD29 and CD44 were high, whereas the expression levels of CD34 and CD45 were low; these findings were consistent with the surface antigen profile of MSCs. (C) Immunofluorescence assays showed clear red fluorescence in postinduction cells (NLCs, NLCs/CMV-FTH1, and NLCs/NSE-FTH1); however, red fluorescence was not evident in preinduction cells (MSCs, MSCs/CMV-FTH1, and MSCs/NSE-FTH1). These results implied that the neuron-specific markers NSE and MAP-2 were not expressed in preinduction cells, but they were clearly expressed in postinduction cells. Scale bars: 25 μm. (D) RT-PCR quantitative analysis revealed obviously higher mRNA expression levels of NSE and MAP-2 in postinduction cells (NLCs, NLCs/CMV-FTH1, and NLCs/NSE-FTH1) than in preinduction cells (MSCs, MSCs/CMV-FTH1, and MSCs/NSE-FTH1). Data are expressed as means ± SDs. Experiments were repeated three times. *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). MAP-2: Microtubule-associated protein-2; MSCs: mesenchymal stem cells; MSCs/CMV-FTH1: ferritin heavy chain 1 overexpression triggered by cytomegalovirus promoter in mesenchymal stem cells; MSCs/NSE-FTH1: ferritin heavy chain 1 overexpression triggered by neuron-specific enolase promoter in mesenchymal stem cells; NLCs: neuron-like cells; NSE: neuron-specific enolase; NLCs/CMV-FTH1: ferritin heavy chain 1 overexpression triggered by cytomegalovirus promoter in neuron-like cells; NLCs/NSE-FTH1: ferritin heavy chain 1 overexpression triggered by neuron-specific enolase promoter in neuron-like cells.
After neuronal induction, the cells became more transparent, and the cytoplasm contracted toward the nucleus. Additionally, most cells exhibited long and thin processes that were connected to adjacent cells. These morphological features were consistent with neuronal morphology (Figure 2A Figure 2C Figure 2D
Specific induction of reporter gene expression by NSE promoter after neuronal differentiation
The mRNA or protein expression of FTH1 in each group of cells was determined by PCR, western blotting, or immunofluorescence staining. PCR revealed remarkably greater mRNA expression of FTH1 in NLCs/NSE-FTH1 than in MSCs/NSE-FTH1. No considerable differences were found in the mRNA expression of FTH1 between MSCs and NLCs or between MSCs/CMV-FTH1 and NLCs/CMV-FTH1 (Figure 3A Figure 3B C NSE promoter specifically induced reporter gene expression after neuronal differentiation, whereas the CMV promoter induced constitutive reporter gene expression (i.e., regardless of neuronal differentiation).
Figure 3: FTH1 expression in cells before and after neuronal differentiation .(A, B) Polymerase chain reaction (A) at the mRNA level and western blotting (B) at the protein level showed remarkably higher expression of FTH1 in NLCs/NSE-FTH1 than in MSCs/NSE-FTH1. However, no considerable differences in FTH1 expression were found between NLCs and MSCs or between NLCs/CMV-FTH1 and MSCs/CMV-FTH1. Data are expressed as means ± SDs. Experiments were repeated three times. *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). (C) Immunofluorescence staining showed remarkably higher FTH1 expression in NLCs/NSE-FTH1 than in MSCs/NSE-FTH1, consistent with the results of polymerase chain reaction and western blotting. The fluorescent indicator for FTH1 was Cy3 (red). Nuclei were stained with DAPI (blue). Scale bars: 25 μm. DAPI: 4′,6-Diamidino-2-phenylindole; FTH1: ferritin heavy chain 1; MSCs: mesenchymal stem cells; MSCs/CMV-FTH1: ferritin heavy chain 1 overexpression triggered by cytomegalovirus promoter in mesenchymal stem cells; MSCs/NSE-FTH1: ferritin heavy chain 1 overexpression triggered by neuron-specific enolase promoter in mesenchymal stem cells; NLCs: neuron-like cells; NSE: neuron-specific enolase; NLCs/CMV-FTH1: ferritin heavy chain 1 overexpression triggered by cytomegalovirus promoter in neuron-like cells; NLCS/NSE-FTH1: ferritin heavy chain 1 overexpression triggered by neuron-specific enolase promoter in neuron-like cells.
Lack of changes in cell viability or differentiation efficiency according to FTH1 expression or iron transport
FAC affected cell viability in a concentration-dependent manner. As the concentration of FAC increased, cell viability decreased; it began rapidly decreasing at a concentration of 200 μM FAC, which was subsequently selected as the optimal concentration of FAC (Figure 4A Figure 4B Figure 4C Figure 4D
Figure 4: Effects of iron on the proliferation and differentiation of MSCs .(A) Cell viability remarkably decreased as the concentration of FAC increased; it began rapidly decreasing at a concentration of 200 μM FAC, which was selected as the optimal concentration of FAC. (B) During 96 hours of culture with 200 μM FAC, no differences in cell proliferation were observed among MSCs, MSCs/CMV-FTH1, and MSCs/NSE-FTH1. (C) There were no significant differences in the rate of neuronal differentiation among the three groups of cells, regardless of FAC treatment. (D) Mean axonal length did not significantly differ among the three groups after neuronal differentiation, regardless of FAC treatment. Data are expressed as means ± SDs. Experiments were repeated three times. *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test [B]; or Student’s t -test [C, D]). FAC: Ferric ammonium citrate; MSCs: mesenchymal stem cells; MSCs/CMV-FTH1: ferritin heavy chain 1 overexpression triggered by cytomegalovirus promoter in mesenchymal stem cells; MSCs/NSE-FTH1: ferritin heavy chain 1 overexpression triggered by neuron-specific enolase promoter in mesenchymal stem cells; OD: optical density.
Changes in cellular MRI contrast according to NSE promoter activity
The MRI signal contrast of MSCs before and after neuronal differentiation was evaluated by T2WI and multi-echo sequences. As shown in Figure 5A B 2 values. However, no differences in T2WI signal intensity or R2 values were found between MSCs and NLCs or between MSCs/CMV-FTH1 and NLCs/CMV-FTH1.
Figure 5: In vitro MRI analysis and iron assay before and after neuronal differentiation .(A, B) The T2WI signal intensity was remarkably lower (A) and the R2 value was remarkably higher (B) in NLCs/NSE-FTH1 than in MSCs/NSE-FTH1. Neither T2WI signal intensity nor R2 values remarkably differed between NLCs and MSCs or between NLCs/CMV-FTH1 and MSCs/CMV-FTH1. (C) Prussian blue staining images revealed numerous intracellular iron particles (blue) in MSCs/CMV-FTH1, NLCs/CMV-FTH1, and NLCs/NSE-FTH1, but not in MSCs, MSCs/NSE-FTH1, or NLCs; these findings were consistent with the results of in vitro MRI analysis. Scale bars: 50 μm. (D) Quantification of intracellular iron showed remarkably higher iron content in NLCs/NSE-FTH1 than in MSCs/NSE-FTH1. However, there were no significant differences between NLCs/CMV-FTH1 and MSCs/CMV-FTH1 or between NLCs and MSCs. Data are expressed as means ± SDs. Experiments were repeated three times. *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc tests). MSCs: Mesenchymal stem cells; MSCs/CMV-FTH1: ferritin heavy chain 1 overexpression triggered by cytomegalovirus promoter in mesenchymal stem cells; MSCs/NSE-FTH1: ferritin heavy chain 1 overexpression triggered by neuron-specific enolase promoter in mesenchymal stem cells; NLCs: neuron-like cells; NLCs/CMV-FTH1: ferritin heavy chain 1 overexpression triggered by cytomegalovirus promoter in neuron-like cells; NLCS/NSE-FTH1: ferritin heavy chain 1 overexpression triggered by neuron-specific enolase promoter in neuron-like cells; R2:transverse relaxation rate.
Accumulation of intracellular iron caused by activation of NSE promoter
The FTH1-induced accumulation of intracellular iron was qualitatively analyzed via Prussian blue staining and quantitively assayed via cellular iron measurements. Prussian blue staining revealed greater accumulation of intracellular iron particles in MSCs/CMV-FTH1, NLCs/CMV-FTH1, and NLCs/NSE-FTH1, compared with MSCs, MSCs/NSE-FTH1, and NLCs, respectively (Figure 5C in vitro MRI findings, indicating that the NSE promoter was activated by neuronal differentiation and therefore induced FTH1 expression, which led to accumulation of intracellular iron. Quantitative analysis revealed remarkably higher intracellular iron content in postinduction cells (NLCs/NSE-FTH1) than in preinduction cells (MSCs/NSE-FTH1). No significant differences were observed between MSCs and NLCs or between MSCs/CMV-FTH1 and NLCs/CMV-FTH1 (Figure 5D
Rat MRI validation
MRI examinations were performed after intracerebral transplantation of the three groups of cells. In the MSCs/CMV-FTH1 group, the T2WI signal intensity remarkably decreased (Figure 6A 2 values did not significantly change between the two time points (Figure 6B Figure 6A 2 values between 3 and 7 days after cell transplantation (Figure 6D
Figure 6: In vivo validation of magnetic resonance imaging for assessment of stem cell differentiation-induced reporter gene expression .(A, B) In the MSCs/CMV-FTH1 group, the T2WI signal intensity remarkably decreased at 3 and 7 days after cell transplantation (A); there was no significant difference in R2 values between the two time points (B). However, in the MSCs/NSE-FTH1 group, the T2WI signal intensity moderately decreased at 3 days but remarkably decreased at 7 days (A); there was a significant difference in R2 values between 3 and 7 days after cell transplantation (B). (C, D) Prussian blue staining revealed extensive accumulation of iron in brain tissue at both 3 and 7 days after cell transplantation in the MSCs/CMV-FTH1 group (C); iron content did not significantly differ between the two time points (D). In the MSCs/NSE-FTH1 group, few iron particles were found in brain tissue at 3 days, whereas numerous iron particles were distributed in brain tissue at 7 days (C). Scale bars: 100 μm. There was a significant difference in tissue iron content between 3 and 7 days after cell transplantation (D). Data are expressed as means ± SDs. *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc tests). MSCs: Mesenchymal stem cells; MSCs/CMV-FTH1: ferritin heavy chain 1 overexpression triggered by cytomegalovirus promoter in mesenchymal stem cells; MSCs/NSE-FTH1: ferritin heavy chain 1 overexpression triggered by neuron-specific enolase promoter in mesenchymal stem cells; NLCs: neuron-like cells; NLCs/CMV-FTH1: ferritin heavy chain 1 overexpression triggered by cytomegalovirus promoter in neuron-like cells; NLCS/NSE-FTH1: ferritin heavy chain 1 overexpression triggered by neuron-specific enolase promoter in neuron-like cells.
Prussian blue staining revealed numerous iron particles distributed in brain tissue at both 3 and 7 days after cell transplantation in the MSCs/CMV-FTH1 group (Figure 6C Figure 6D Figure 6C Figure 6D NSE promoter-driven FTH1 expression was dependent on neuronal differentiation, whereas CMV-driven reporter gene expression could not indicate whether cells had undergone differentiation.
Discussion
In stem cell-based therapies, transplanted cell monitoring is necessary to understand cell fate (James et al., 2021; Zhang et al., 2021). In this study, we sought to noninvasively detect neuronal differentiation in stem cells; we developed an activatable reporter gene system in which the neuron-specific promoter NSE acted as a genetic switch that controlled the expression of FTH1. This system allowed cell type-dependent expression of the reporter gene, whereby the reporter gene was initially silenced but became activated when the cells underwent neuronal differentiation. To our knowledge, this is the first study to utilize NSE as a promoter to induce FTH1 expression and facilitate MRI-based monitoring of neuronal differentiation in MSCs. Our findings indicated that FTH1 expression was remarkably increased after stem cells had undergone neuronal differentiation; the resulting intracellular iron content was sufficient for detection by MRI. In combination with this novel reporter gene system, MRI can be used to determine whether and/or when cell differentiation occurs after the original stem cells are transplanted; this approach will facilitate broader implementation of stem cell replacement therapies.
In the past 10 years, there has been increasing evidence that MSCs can differentiate into neuronal cells in a particular biological environment (Bi et al., 2014; Mu et al., 2018; Shokati et al., 2021; Rahmani-Moghadam et al., 2022). Various strategies have been used to induce stem cells to undergo neuronal differentiation (Bi et al., 2014; Shokati et al., 2021; Rahmani-Moghadam et al., 2022). In this study, to improve neuronal differentiation efficiency, MSCs were pretreated with 1 mmol/mL ATRA for 24 hours and then cultured with modified neuronal induction medium. After this two-step neuronal induction method, the induced cells exhibited a morphology similar to neuronal morphology; they also expressed particularly high levels of NSE and MAP-2, consistent with previously reports (Bi et al., 2010; Mu et al., 2018). Although the induced cells were similar to neurons in terms of morphology and some cellular markers, they remain distinct from true neurons. Thus, we regard them as NLCs. Although these induced cells can promote functional recovery in rats with brain injury (Liu et al., 2008), their true functions in vivo and their abilities to establish reciprocal connections with host neurons remain unknown. In the present study, after pretreated MSCs had been transplanted into rat striatum for 3 days, Prussian blue staining revealed accumulation of intracellular iron, which may be a result of NSE expression; this finding indicated that the grafted cells had begun to differentiate into NLCs. The duration of time needed for in vivo differentiation differed from the duration of time needed for in vitro differentiation. This difference may be attributed to differences in the environments in which the cells were induced.
Cell differentiation involves the selective expression of genes in time and space, where the expression of different genes is switched on or off, with effects on the production of marker proteins (Li et al., 2022; Wang et al., 2022). Some specific promoters are closely involved in cell differentiation processes and play key roles in determining cell lineage commitment by controlling the expression patterns of specific genes. In this context, a tissue-specific promoter could be used for specific induction of reporter gene expression in cells or tissues of interest. In this study, we sought to achieve real-time monitoring of neuronal differentiation in stem cells; this required identifying a gene that was specifically expressed in neuronal cells, then using its promoter to induce reporter gene expression. Genes specifically expressed in neuron cells include synaptic protein 1 (synapsin), doublecortin, and NSE (Hwang et al., 2008; Pohlkamp et al., 2014; Tennstaedt et al., 2015; Yun et al., 2018). As a protein present in synaptic vesicles, synaptic protein 1 is upregulated in mature neurons. It is nearly undetectable in the initial phase of neuronal differentiation in stem cells, which suggests that the use of a synaptic protein 1-driven reporter gene may not enable the early detection of neuronal differentiation in stem cells (Tennstaedt et al., 2015). Doublecortin is a specific marker in the early phase of neuronal differentiation, but it is nearly undetectable in differentiated mature neurons. Therefore, the doublecortin promoter cannot drive reporter gene expression in mature neuronal cells (Tennstaedt et al., 2015). NSE is highly active in both immature and mature nerve cells (Pohlkamp et al., 2014); its promoter is ideal for use in a reporter gene imaging strategy to achieve noninvasive detection of neuronal differentiation. In the present study, the NSE promoter was used to control FTH1 expression, then compared with expression under the CMV promoter, during neuronal differentiation in stem cells. Our results indicated that FTH1 expression was remarkably higher in NLCs/NSE-FTH1 than in MSCs/NSE-FTH1; it was also comparable between NLCs/NSE-FTH1 and NLCs/CMV-FTH1, suggesting that similar activation of FTH1 expression could be achieved by the NSE promoter and the CMV promoter during neuronal differentiation in stem cells.
For the tissue-specific reporter imaging strategy, changes in the imaging signal were determined by promoter activity. Our results showed clear changes in the MRI signal after stem cells had undergone neuronal differentiation. In vitro analyses showed a significant decrease in the MRI signal in NLCs/NSE-FTH1, compared with MSCs/NSE-FTH1, consistent with the results of western blotting and Prussian blue staining. However, the MRI signal did not differ between NLCs/CMV-FTH1 and MSCs/CMV-FTH1. These results demonstrated that FTH1 expression was successfully induced by the NSE promoter, which is only activated in cells that have undergone neuronal differentiation. For in vivo analyses, transgenic MSCs pretreated with ATRA were engrafted into the rat striatum. In the MSCs/CMV-FTH1 group, the MRI signal was remarkably lower than the signal in the control group, and no difference was found between 3 and 7 days after cell transplantation. However, in the MSCs/NSE-FTH1 group, the MRI signal intensity moderately decreased at 3 days but remarkably decreased at 7 days after cell transplantation. These differences in signal dynamics between the two groups could be attributed to the NSE activation status. In the MSCs/CMV-FTH1 group, the CMV promoter was not influenced by cell differentiation status, and the reporter gene was constitutively expressed regardless of cell differentiation. However, in the MSCs/NSE-FTH1 group, FTH1 expression was driven by the NSE promoter; its activity was influenced by cell differentiation status. The difference in the MRI signal between 3 and 7 days in the MSCs/NSE-FTH1 group could be the result of differences in NSE activation during the progression of neuronal differentiation.
Although the findings provided preliminary confirmation of the feasibility of MRI-based detection of neuronal differentiation by promoter-driven reporter gene expression, this study had some limitations. First, to confirm the effect of reporter gene expression on intracellular iron transport, we added a specific concentration of FAC to cells or tissues in vitro and in vivo ; this provided a sufficient source of iron for intracellular iron uptake, thereby enabling MRI contrast generation. Such an artificial iron environment can be used in animal experiments to compensate for the low sensitivity of iron detection by MRI, but it is not appropriate for clinical applications of reporter gene imaging strategies (He et al., 2015; Sun et al., 2021). As new technologies become available, we expect that new-generation MRI will have sufficient sensitivity to detect the accumulation of intracellular iron caused by reporter gene expression in the context of endogenous iron concentrations; this sensitivity should be investigated in future studies. Second, because the main purpose of our in vivo experiment was to confirm the feasibility of MRI-based detection of stem cell differentiation-induced reporter gene expression, and because of animal welfare considerations, we did not conduct additional assessments of stem cell fate. Future longitudinal studies are needed to assess the differentiation of grafted cells into NLCs, the survival of those cells, and the ability of those cells to integrate into (and function within) normal neural tissue. Third, we only used a two-step method to induce MSC differentiation into NLCs; in this method, the NSE promoter was used to initiate reporter gene expression. However, in clinical applications, stem cells from diverse sources may differentiate into nerve cells with various functions, such as cholinergic and dopaminergic cells (Tantrawatpan et al., 2013; Kirkeby et al., 2017; Han and Hu, 2020; Stricker et al., 2021); other cell-specific promoters will be needed to drive the reporter gene expression in those cells. Finally, only male rats were used in the in vivo experiment to maintain consistency between in vivo and in vitro experiments (we extracted MSCs from male rats for the in vitro analysis), and to reduce immune rejection after cell transplantation. Future studies should use female rats to determine whether this reporter system is influenced by sex-related differences.
In conclusion, we established a reporter system in which expression of the FTH1 gene was controlled by a neuron-specific promoter, then used the system to confirm the feasibility of MRI-based detection of neuronal differentiation in stem cells. In this reporter system, the reporter gene was initially silenced but became activated when the cells underwent neuronal differentiation. This differential expression allowed differentiation-based activation of the reporter gene and enabled MRI to sensitively detect the onset of neuronal differentiation in stem cells. This neuron-specific MRI monitoring system could facilitate broader use of stem cell-based therapeutic strategies by noninvasively indicating whether and/or when stem cells undergo neuronal differentiation.
Acknowledgments: We are grateful to the Key Laboratory of Pediatrics in Chongqing for providing parts of the experimental apparatus .
Author contributions: Study conception and design: JHC; sample preparation and processing: XYH, YRZ, YFL; cell preparation: XYH, YRZ, TM, YFL; data acquisition, analysis, and interpretation: XYH, YRZ, TM, YFL; statistical analysis: XYH, YRZ, TM; manuscript drafting: XYH, YRZ; experiment study: XYH, YRZ, TM, YFL, YQ, LJ; study supervision: JHC. All authors reviewed and approved the final version of the manuscript .
Conflicts of interest: The authors have no conflicts of interest to disclose .
Availability of data and materials: All data generated or analyzed during this study are included in this published article and its supplementary information files .
Additional file:
Additional file 1 : The sequences of the FTH1 gene and NSE promoter .
C-Editor: Zhao M; S-Editors: Yu J, Li CH; L-Editors: Chastain-Gross R, Yu J, Song LP;
T-Editor: Jia Y
Funding: This study was supported by the National Natural Science Foundation of China, No. 81771892 (to JHC) .
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